Variant DNA Polymerases Having Improved Properties and Method for Improved Isothermal Amplification of a Target DNA

ABSTRACT

Variants of the bacteriophage B103 DNA polymerase are described herein. The variant has improved properties, that include when compared to wild-type Phi29 DNA polymerase, at least one of the following: increased thermostability, improved reaction rate for DNA amplification, reduced background and a reduction of bias. Methods of using the DNA polymerase variant are also described herein.

CROSS REFERENCE

This application is a divisional application of U.S. application Ser.No. 16/598,554, filed Oct. 10, 2019, which claims priority to U.S.Provisional Application No. 62/743,718, filed Oct. 10, 2018, hereinincorporated by reference.

BACKGROUND

Members of the Phi29 family of DNA polymerases, such as the polymerasesfrom bacteriophage Phi29, B103, M2(Y) and Nf, do not need accessoryproteins and possess a number of distinctive biochemical propertiesincluding a strong binding capacity for single stranded DNA, stranddisplacement activity, high processivity and a proofreading activity(see, e.g., Meijer et al Microbiol Mol Biol Rev. 65: 261-287 (2001)).These properties make these DNA polymerases suitable in a variety ofapplications, including rolling circle amplification (RCA), multiplestrand displacement amplification (MDA) and whole genome amplification(WGA) (see, e.g., Fakruddin et al, J Pharm Bioallied Sci. 5: 245-252(2013)). The DNA polymerase of bacteriophage Phi29 from Bacillussubtilis is a well-studied example of this class of polymerases (see,e.g., Garmendia et al, Journal of Biological Chemistry, 267, 2594-2599(1992) and Esteban et al Journal of Biological Chemistry, 268, 2719-2726(1993)).

There is a continued need for modified polymerases that have improvedproperties with respect to amplifying DNA. Desirable improved propertiesmay include any one or more of the following: improved efficiency ofpolymerase activity, specificity and accuracy, yield, reduced sequencebias, reduction in non-template amplification; and capability of workingwell at elevated temperatures and/or high salt conditions.

SUMMARY

In one embodiment, the composition has (a) an amino acid sequence thathas at least 95% sequence identity with SEQ ID NO:2; and (b) one or moreamino acid substitutions at positions selected from the group consistingof positions 147, 221, 318, 339, 359, 372, 503, 511, 544, 550, wherein,in the polymerase: the amino acid at a position 147 is not alanine orthreonine, the amino acid at a position 221 is not arginine or lysine,the amino acid at a position 318 is not alanine or valine, the aminoacid at a position 339 is not methionine, the amino acid at a position359 is not glutamic acid, the amino acid at a position 372 is notlysine, the amino acid at a position 503 is not alanine or valine, theamino acid at a position 511 is not isoleucine or lysine, the amino acidat a position 544 is not alanine or arginine, and the amino acid at aposition 550 is not methionine or threonine, wherein the positionscorrespond to positions of SEQ ID NO:2.

In other embodiments, the composition includes (a) an amino acidsequence that has at least 95% sequence identity with SEQ ID NO:2; and(b) at least one, two, three, four, five, six or all seven of thefollowing amino acid substitutions selected from the group consistingof: a lysine that replaces alanine at position 147, a tyrosine thatreplaces arginine at position 221, a glycine that replaces alanine atposition 318, methionine that replaces alanine at position 503, valinethat replaces isoleucine at position 511, lysine that replaces anarginine at position 544, and lysine that replaces threonine at position550. Additional substitutions may occur at any or all of positions 339,359, 383 and 384. Alternatively, the composition has at least 99%sequence identity with SEQ ID NO:5. In these embodiments, thecomposition has one or more improved DNA polymerase properties relativeto wild-type Phi29 DNA polymerase tested under the same experimentalconditions. The improved properties may include one or more of: anincreased thermostability, for example at temperatures of 30° C.-45° C.;an increased reaction rate for DNA amplification over a defined period(e.g. 120 minutes or less), wherein the increase is at least 10%; agreater yield of amplified DNA; a reduction in background amplificationarising from artefacts (nonspecific amplification in the absence oftemplate); a reduction of bias that causes under-representation of GCrich regions and over-representation of AT rich regions; and a reductionin undesirable chimeric amplicons such as formed from primer-templatefusions, non-sequential template sequences, or primer-non-sequentialtemplate sequences.

In any of the embodiments described above, the composition may be afusion protein. The composition may be in a mixture of the variants withother types of polymerases and/or other enzymes with different substratespecificities.

In another aspect, the composition may include a buffering agent and mayfurther include: dNTPs and/or modified dNTPs and/or one or more primers.

In one embodiment, a kit is provided that contains: (i) a DNA polymeraseof the type described above; and (ii) a buffer. The kit may contain theDNA polymerase in a lyophilized form or in a storage buffer and/or withthe reaction buffer in concentrated form. The kit may contain the DNApolymerase in a mastermix suitable for receiving template nucleic acidfor causing amplification to occur. The DNA polymerase may be a purifiedenzyme so as to contain substantially no DNA or RNA and no nucleases.The reaction buffer in (ii) and/or storage buffers containing the DNApolymerase in (i) may include nonionic, ionic e.g. anionic orzwitterionic surfactants and crowding agents. The kit may additionallycontain one or more primers such as for example, random primers,exonuclease-resistant primers or primers having chemical modifications.The kit may further include one or more dNTPs including those dNTPs withlarge adducts such as a fluorescent-label or biotin-modified nucleotide,or a methylated nucleotide or other modified nucleotide. The kit mayinclude the DMA polymerase and reaction buffer in a single tube or indifferent tubes.

In one aspect, a DNA encoding a DNA polymerase such as described aboveis provided. In another aspect, a host cell containing DNA for encodingthe above described DNA polymerase is provided.

In general, a method for isothermal amplification of a target DNA isprovided that includes combining the target DNA with a DNA polymerase ofthe type described above, dNTPs or modifications thereof and,optionally, one or more primers, to produce a reaction mixture; andincubating the reaction mixture to amplify the target DNA. Theamplification reaction may be performed at a temperature in the range of30° C.-42° C. and optionally a high salt buffer. The target DNA may be awhole genome or a target DNA sequence therein. The target DNA may belinear or circular.

In one aspect, the isothermal amplification of the target DNA isachieved using a DNA polymerase variant that includes for example (1)(a) an amino acid sequence having at least 95% sequence identity withSEQ ID NO:2; and (b) at least one, two, three, four, five, six or allseven of the following amino acid substitutions selected from the groupconsisting of: a lysine that replaces alanine at position 147, atyrosine that replaces arginine at position 221, a glycine that replacesalanine at position 318, methionine that replaces alanine at position503, valine that replaces isoleucine at position 511, lysine thatreplaces an arginine at position 544, and lysine that replaces threonineat position 550. Additional substitutions may occur at any or all ofpositions 339, 359, 383 and 384; or (2) at least 99% sequence identitywith SEQ ID NO:5. Using the same molar ratio of polymerase to substratewhere the target DNA is either linear or circular, one or more improvedproperties relative to wild-type Phi29 DNA polymerase was achieved. Theimproved properties include one or more of the following: an increasedthermostability; an increased reaction rate for DNA amplification,wherein the increase may be at least 10%; increased yield of amplifiedDNA; reducing background amplification arising from artefacts; andreduced bias against GC rich regions and in favor of AT rich regions, toprovide an even coverage of the substrate DNA.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1A-1D shows that a variant of the B103 DNA polymerase (referred toas “mutant” in the figures) that has the amino acid sequence of SEQ IDNO:5 generates amplification products by RCA and WGA with higher yield(RFU) and in a shorter time than wild-type Phi29 polymerase at alltemperatures tested. As shown here, wild-type Phi29 is not active above30° C., whereas the variant is active at 42° C.

FIG. 1A shows that the variant DNA polymerase is not only capable of RCAat 37.6° C. and 42° C. as well as at 30° C. but it actually performssignificantly better at the higher temperatures producing yields(indicated by the RFU on the y-axis, reflective of the amount of DNAproduced in the reaction) that are close to maximum at 60 minutes.Moreover, the yields are significantly higher than observed for thewild-type Phi29 DNA polymerase used as a control.

FIG. 1B shows that wild-type Phi29 polymerase is active here only up to30° C. and not at the higher temperatures tested. Moreover, yields fromthis reaction did not approach their maximum until at least 220 minutesof incubation.

FIG. 1C shows that the variant DNA polymerase is not only capable of WGAat 37.3° C. and 41.5° C. as well as at 30° C. and produced higher yieldsof products at the higher temperatures. For example, the time taken forthe variant DNA polymerase to produce a yield of 4000 RFU at 41.5° C.was 26 minutes. The time taken for the mutant to produce the same amountof product at 37.5° C. was 27 minutes, whereas at 30° C. the time takenwas 58 minutes.

FIG. 1D shows that the wild-type Phi29 polymerase control was not activeat 37.3° C. nor at 41.5° C., and at 30° C. it took 87 minutes to reach4000 RFU. The maximum yield was similar to the variant DNA polymerase at30° C.

FIG. 2A-2B shows that variant DNA polymerase provides an increased yieldof RCA amplification product at temperatures above 32° C. up to andincluding 42° C. compared with the wild-type Phi29 DNA polymerase.

FIG. 2A shows that the variant DNA polymerase produces a high yield ofamplified DNA at all temperatures tested: 30° C., 30.7° C., 32.3° C.,34.6° C., 37.6° C., 39.9° C., 41.3° C. and 42° C. However, yieldsincreased significantly at 34.6° C., 37.6° C., 39.9° C., 41.3° C. and42° C.

FIG. 2B shows that the wild-type Phi29 polymerase control only producedamplified DNA at 30° C., 30.7° C., 32.3° C., 34.6° C. with an end pointyield comparable to the variant DNA polymerase at low temperatures,after which no product was detected.

FIG. 3A-3D shows that use of the variant DNA polymerase increases theyield of amplification product in WGA and reduces backgroundamplification at temperatures above 30° C. compared with the controlPhi29 DNA polymerase.

FIG. 3A shows the increase in yield of DNA (ng/μl) using WGA with thevariant DNA polymerase at all temperatures tested.

FIG. 3B shows the yield of DNA (ng/μl) using WGA with the controlwild-type Phi29 DNA polymerase at the same temperatures as FIG. 3A wherethe yield is significantly less at 30° C.

FIG. 3C shows the results of qPCR analysis of amplified target overtotal DNA (1 ng Hela genomic DNA) showing that the variant DNApolymerase (at 41.5° C.) generated a higher fraction of the target DNAwith less undesirable background amplification of primer sequencescompared with the control Phi29 polymerase (at 30° C.). The amount andevenness of specifically amplified DNA (as opposed to non-specificamplification products and biased amplification) in the amplifiedproduct was determined by qPCR using 4 pairs of primers for target DNAsequences located on different chromosomes (3p, 4p, 7p and 13p). Theamount of specifically amplified DNA was calculated as a fraction of thetotal DNA quantified by PicoGreen® (Molecular Probes, Eugene, Oreg.).

FIG. 3D shows that total yield of amplified DNA using the variant DNApolymerase is 5000-fold and with the wild-type is 3500-fold whendetected using PicoGreen, under the conditions used in FIG. 3C with 1 nginput genomic DNA (gDNA) for amplification.

FIG. 4 shows that WGA with the mutant polymerase can be conducted atelevated temperature (to 42° C.) with high yield of amplificationproduct and reduction of non-specific amplification. The no templatecontrol (NTC) reaction produces high yield of DNA product at lowertemperatures, but at temperatures of greater than 41° C. the NTCreaction is substantially suppressed while the positive reactioncontinues to produce high yield.

The solid line (circles) shows reactions using 1 ng of DNA template(Hela DNA) and variant DNA polymerase. The dotted line (triangles) is anidentical reaction with primers but lacking DNA template (NTC).

DNA was quantified using PicoGreen in this experiment.

FIG. 5 shows the mutant polymerase produces amplified DNA thatrepresents even coverage of gDNA template as measured via DNAsequencing. Examples of the mutants used here are Mutant 1 (SEQ ID NO:5)and Mutant 2 (SEQ ID NO:6)

FIG. 6 shows an alignment of selected wild-type and mutant polymerasesthat share the properties of the polymerases described herein.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, NewYork (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with thegeneral meaning of many of the terms used herein. Still, certain termsare defined below for the sake of clarity and ease of reference.Preferably, any further interpretations of terms should be consistentwith U.S. Pat. No. 10,034,951.

As used herein, the term “DNA polymerase” refers to an enzyme that iscapable of replicating DNA. The enzyme may also have reversetranscriptase properties. Examples of polymerases from the phage Phi29family include polymerases from phages Phi29, B103, GA-1, PZA, Phi15,BS32, M2(Y) and Nf (Microbiology and Molecular Biology Reviews, 65(2),261-286 (2001)).

As used herein, the term “DNA polymerase variant” and “DNA polymerasemutant” refers to a non-naturally occurring bacteriophage DNA polymerasethat has an amino acid sequence less than 100% identical to the aminoacid sequence of a wild-type DNA polymerase from Phi29 family of phages.A variant amino acid sequence may have at least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, at least 98% or at least 99%identity to the naturally occurring amino acid sequence. Sequencedifferences may include insertions, deletions and amino acidsubstitutions.

A mutant or variant protein may be a “fusion” protein. The term “fusionprotein” refers to a protein composed of a plurality of polypeptidecomponents that are un-joined in their native state. Fusion proteins maybe a combination of two, three or even four or more different proteins.The term polypeptide is not intended to be limited to a fusion of twoheterologous amino acid sequences. A fusion protein may have one or moreheterologous domains added to the N-terminus, C-terminus, and or themiddle portion of the protein. If two parts of a fusion protein are“heterologous”, they are not part of the same protein in its naturalstate.

Examples of fusion proteins includes a Phi29 variant fused to an SSO7DNA binding peptide (see for example, U.S. Pat. No. 6,627,424), atranscription factor (see for example, U.S. Pat. No. 10,041,051), abinding protein suitable for immobilization such as maltose bindingdomain (MBP), chitin binding domain (CBD) or SNAP-Tag® (New EnglandBiolabs, Ipswich, Mass. (see for example U.S. Pat. Nos. 7,939,284 and7,888,090). The binding peptide may be used to improve solubility oryield of the polymerase variant during the production of the proteinreagent. Other examples of fusion proteins include a heterologoustargeting sequence, a linker, an epitope tag, a detectable fusionpartner, such as a fluorescent protein, (3-galactosidase, luciferase andthe functionally similar peptides.

As used herein, the term “buffering agent”, refers to an agent thatallows a solution to resist changes in pH when acid or alkali is addedto the solution. Examples of suitable non-naturally occurring bufferingagents that may be used in the compositions, kits, and methods of theinvention include, for example, any of Tris, HEPES, TAPS, MOPS, tricine,and MES.

The buffering agent which may be combined with the polymerase variant,may further include additional reagents such as crowding agents (such aspolyethylene glycol included in the storage and/or reaction mixture),single strand binding proteins or portions thereof, unwinding agentssuch as helicases, detergents such as nonionic, cationic, anionic orzwitterionic detergents, additives such as albumin, glycerol, salt (e.g.KCl), reducing agent, EDTA, dyes, a reaction enhancer or inhibitor, anoxidizing agent, a reducing agent, a solvent and/or a preservative. Thebuffering agent combined with additional reagents that are standard inthe art may be formulated for storage of the Phi29 variant or for thereaction mixture. The formulation for storage of the polymerase variantor for an amplification reaction may be the same or different.

The polymerase may be prepared for storage as a reagent or contained ina mastermix for storage. Alternatively, the polymerase may be containedin a reaction buffer or mastermix. The reaction mix and the storage mixmay be the same or different. The storage mix may be a concentrated formof the reagent for dilution into a reaction mix. In one example, thepolymerase variant may be in a lyophilized form. In another example, thepolymerase variant may be in a master mix containing deoxyribosenucleoside triphosphates e.g. one, two, three or all four of dATP, dTTP,dGTP and dCTP and/or one or more modified dNTPs. The composition mayoptionally comprise primers. In some embodiments, the primers may bepartially or complete random. In some embodiments, the primers may beexonuclease-resistant. In some embodiments, primers may comprise one ormore chemical modifications, for example, phosphorothioatemodifications. In some embodiments, the chemical modifications on theprimers may occur at 3′-terminal or both of 3′ and 5′ terminals of theprimers. In some embodiments, the chemical modifications on the primersmay further occur at one or more non-terminal positions of the primers.

The term “non-naturally occurring” refers to a composition that does notexist in nature.

A “non-naturally occurring” protein may have an amino acid sequence thatis different from a naturally occurring amino acid sequence for example,one or more amino acid substitutions, deletions or insertions at theN-terminus, the C-terminus and/or between the N- and C-termini of theprotein. Hence the non-naturally occurring protein may have less than100% sequence identity to the amino acid sequence of a naturallyoccurring protein although it may have least 80%, at least 85%, at least90%, at least 95%, at least 97%, at least 98%, at least 98.5% or atleast 99% identical to the naturally occurring amino acid sequence. Incertain cases, a non-naturally occurring protein may include a proteinthat has a post-translational modification pattern that is differentfrom the protein in its natural state for example, an N-terminalmethionine or may lack one or more post-translational modifications(e.g., glycosylation, phosphorylation, etc.) if it is produced by adifferent (e.g., bacterial) cell.

In the context of a nucleic acid, the term “non-naturally occurring”refers to a nucleic acid that contains: a) a sequence of nucleotidesthat is different from a nucleic acid in its natural state (i.e., havingless than 100% sequence identity to a naturally occurring nucleic acidsequence); b) one or more non-naturally occurring nucleotide monomers(which may result in a non-natural backbone or sugar that is not G, A, Tor C); and/or c) may contain one or more other modifications (e.g., anadded label or other moiety) to the 5′-end, the 3′ end, and/or betweenthe 5′- and 3′-ends of the nucleic acid.

In the context of a preparation, the term “non-naturally occurring”refers to: a) a combination of components that are not combined bynature, e.g., because they are at different locations, in differentcells or different cell compartments; b) a combination of componentsthat have relative concentrations that are not found in nature; c) acombination that lacks something that is usually associated with one ofthe components in nature; d) a combination that is in a form that is notfound in nature, e.g., dried, freeze dried, crystalline, aqueous; and/ore) a combination that contains a component that is not found in nature.For example, a preparation may contain a “non-naturally occurring”buffering agent (e.g., Tris, HEPES, TAPS, MOPS, tricine or MES), adetergent, a dye, a reaction enhancer or inhibitor, an oxidizing agent,a reducing agent, a solvent or a preservative that is not found innature.

The non-naturally occurring polymerase may be purified so that it doesnot contain DNases, RNases or other proteins with undesirable enzymeactivity or undesirable small molecules that could adversely affect thesample substrate or reaction kinetics.

The term “corresponding to” in the context of corresponding positions,refers to positions that lie across from one another when sequences arealigned, e.g., by the BLAST algorithm.

DNA Polymerase Variants and Compositions Containing the Same

Provided herein is a Phi29 like DNA polymerase identified asbacteriophage B103 DNA polymerase (SEQ ID NO:2), M2(Y) DNA polymerase(SEQ ID NO:3) and Nf DNA polymerase (SEQ ID NO:4). Non-natural variantsof B103 are described by SEQ ID NOs:5-27.

The examples demonstrate the assay for and detection of improvedproperties using one of the variants (SEQ ID NO:5). In some embodiments,the variant has an amino acid sequence that has at least 98%, at least98.5% or at least 99% identical to SEQ ID NO:2. For example, the variantmay have one or more (e.g., one, two, three, four, five or more thanfive) amino acid substitutions at positions from the group consisting of73, 147, 221, 318, 503, 511, 544, 550. Examples of one or more, two ormore, three of more, four or more, five or more, six or more or seven ormore mutations at these sites include: H73R, A147K, R221Y, A318G, A503M,I511V, R544K, T550K. Additionally, one or more substitutions may beselected from position 339, 359, 372, 383 and 384. Alternatively, when avariant has a mutation at position 339, 359, 372, 383 and/or 384, italso has at least one mutation selected from: H73R, A147K, R221Y, A318G,A503M, I511V, R544K, and T550K. In some embodiments, the variant mayhave an amino acid sequence that is at least 98.5%, at least 99% or 100%identity to the amino acid sequence of SEQ ID NO:5.

In some embodiments, the variant DNA polymerase may have an amino acidsequence that is at least 80%, or at least 90% identical to SEQ ID NO:1(i.e., the wild-type Phi29 DNA polymerase). In some embodiments, thevariant DNA polymerase may have one or more substitutions at positionsselected from the group consisting of: V16, E17, N28, E30, D31, H32,S33, E34, A46, L49, K50, I68, N74, A80, D81, R93, L104, I112, T137,V138, Y153, K154, A161, Q168, E172, L175, I199, T200, K205, T210, G214,V219, R233, F234, L259, E264, E269, V273, W274, D277, H284, C287, R303,S304, R305, Y307, S315, I320, A321, D322, W324, S326, M333, K334, D338,S346, L348, K351, A352, T353, I367, T370, S371, N406, A408, L413, E415,T418, Y436, I464, D466, V467, G513, D517, Y518, D520, I521, K533, E537,E541, R549, M551, P559, D567, T568 corresponding to positions of SEQ IDNO:1.

In some embodiments, the DNA polymerase variant may comprise an aminoacid sequence that is at least 80% or at least 90% identical to SEQ IDNO:1, it may further comprise one or more amino acid substitutionselected from the group consisting of: V16L, E17D, N28E, E30G, D31N,H32L, S33D, E34N, A46Q, L49M, K50E, 168V, N74H, A80N, D81E, R93K, L104F,I112L, T137P, V138L, Y153H, K154E, A161E, Q168E, E172R, L175D, I199L,T200S, K205N, T210K, G214P, V219I, R233K, F234Y, L259P, E264A, E269QV273E, W274K, D277Q, H284R, C287F, R303K, S304N, R305P, Y307F, S315N,I320P, A321V, D322E, W324Y, S326T, M333I, K334Q, D338E, S346D, L348F,K351R, A352E, T353K, 1367V, T370H, S371E, N406D, A408S, L413V, E415D,T418Y, Y436F, 1464V, D466E, V467I, G513C, D517E, Y518A, D520T, I521T,K533T, E537K, E541D, R549S, M551G, P559N, D567S, T568V corresponding topositions of SEQ ID NO:1.

In some embodiments, the DNA polymerase variant may have an amino acidsequence that is at least 95% identical to SEQ ID NO:3 (the wild-typeM2(Y) DNA polymerase) and may further comprise at least one substitutionat positions corresponding to SEQ ID NO:3, wherein the position isselected from the group consisting of: S2, Q73, T147, K221, V318, V503,K511, A544, M550. These substitutions may further comprise: S2P, Q73R,T147K, K221Y, V318G, V503M, K511V, A544K, and/or M550K. In oneembodiment, the variant may optionally lack any mutations at positions359, 372, 383 and 384.

In some embodiments, the DNA polymerase variant may have an amino acidsequence that is at least 95% identical to SEQ ID NO:4 (wild-type Nf DNApolymerase) and may further comprise at least one substitution atpositions corresponding to SEQ ID NO:4, wherein the position is selectedfrom the group consisting of: S2, Q73, R107, T147, K221, V318, V503,K511, A544, M550. These substitutions may further comprise: S2P, Q73R,T147K, K221Y, V318G, V503M, K511V, A544K, and/or M550K. In oneembodiment, the variant may optionally lack any mutations at positions359, 372, 383 and 384.

Examples of reaction conditions are provided in the examples andfigures. Providing incubation of the amplification reaction is permittedto occur for an extend period of time (e.g. 4 hours), the yield ofamplified DNA reaches a plateau regardless of whether the DNA polymeraseis a Phi29 family variant or wild type. However, with the variants, therate of reaction is more rapid resulting in shorter incubation times toreach approximately maximum yield. The thermostability of the variantmay contribute to the reduction of bias that occurs from high or low GCcontent. Hence the variant polymerase are more efficient at amplifyingtemplate DNA than the wild type phi29 under the same conditions.

The DNA polymerase variants described herein show improved propertiescompared to wild-type Phi29 DNA polymerase under the same conditions asexemplified by Mutant 1. Suitable variants preferably have increasedpolymerase activity at a temperature range of 30° C. to 45° C. relativeto a wild-type Phi29 DNA polymerase and increased thermostability atleast within a temperature range of 35° C.-42° C. Examples of suitabletemperature ranges for the variants may include for example, 35° C. to40° C., 40° C. to 45° C. or 35° C. to 50° C.

DNA polymerase variants described herein showed the improved propertiesof thermostability, reduction in template independent products,increased overall yield of amplified product, and higher amplificationefficiency, Increased thermostability of the variants at elevatedtemperature provides an increase in amplification product with reductionin DNA sequence bias as a measure of the efficiency of the reaction overa selected time period (e.g. 100 minutes or 120 minutes). The increasein yield of amplification product for exemplified time periods may be atleast 10% more, at least 20% more, at least 50% more, at least 100%more, at least 200% or at least 250% compared with the wild-type Phi29DNA polymerase under the same conditions.

The significant increases in yield achieved with the variants is shownin FIGS. 1A-1D, 2A-2B and 3A-3D where the thermostability of selectedvariants (e.g. Mutant 1) at elevated temperatures permits a significantincrease in the yield of amplification products of target DNA during aselected time period of the reaction (for example, within the first 120minutes) where the yield at 41.5° C. and 37.5° C. was compared to 30° C.using either RCA or WGA amplification methods. Similar results areexpected for other isothermal amplification methods amenable to the useof the Phi29 family of polymerases.

FIG. 3C and FIG. 5 and the Tables 1-4 provided evidence that thevariants could reduce amplification bias against high and low GC regionsand reduce chimera formation in amplified DNA. The use of the DNApolymerase variants at an elevated temperature reduced the backgroundresulting from generation of undesired template-independent DNA in anamplification reaction to a negligible level. Taken together, theresults show that at least those polymerase variants that have at least98.5% or 99% sequence identity to SEQ ID NO:5 or at least 95% sequenceidentity with SEQ ID NO:2 with at least one, two, three, four, five, sixor all amino acid substitution selected from the group consisting of:A147K, R221Y, A318G, A503M, I511V, R544K and T550K have both an improvedrate of reaction leading to at least the same or higher maximum yield ofproduct DNA with less bias against high GC or low GC template DNA. Thiscombination is referred to as the improved efficiency of amplificationwith the variants. An additional benefit of the use of these variantpolymerases is the significant reduction to negligible levels onnon-template amplification (see FIG. 4).

The DNA variants described herein show reduced bias in amplification ofsubstrate DNA so that WGA of a genome provided an even and unbiasedamplification of all regions of varying GC content as determined bysequencing that was sequence platform independent (see for example, FIG.5).

For example, Table 1 shows that the DNA polymerase variant has animproved ability to evenly amplify mixed genomic DNAs of varying GCcontent including Rhodopseudomonas palustris (65% GC content) andHaemophilus influenzae (38% GC). Formulations of wild-type Phi29produced very few sequencing reads from R. palustris genome while a highpercentage of the total reads were represented by the H. influenzaegenome. In contrast, the variants described herein produced sequencingreads from the high GC gDNA that matched the mixed pool demonstratingmore even and less biased amplification of the mixed pool. Table 2 showsthat the DNA polymerase variant produced significantly fewer chimericsequencing reads in a long-read nanopore sequencing experiment than didthe wild-type Phi29 where the chimera may be formed by amplification oftarget DNA or in a non-sequential manner due to annealing and folding ofthe target genomic DNA.

In embodiments, a method is provided for isothermal amplification oftarget DNA using a DNA polymerase variant as described herein.Accordingly, the target DNA is combined with the variant DNA polymeraseand dNTPs to produce a reaction mixture preferably further including oneor more primers; and the reaction mixture incubated to amplify thetarget DNA.

Accordingly, the DNA polymerase variants were demonstrated to provideimproved results with strand displacement amplification, RCA and MDA.For example, the DNA polymerase variants were used in conjunction withrandom primers to amplify circularized DNA by RCA to produce high yieldsof product (for an explanation of RCA see, e.g., Dean et al., GenomeRes. 11:1095-1099 (2001)). For RCA, oligonucleotide primers that arecomplementary to the target circle of DNA hybridize to the sample. Theaddition of DNA polymerase and deoxynucleoside triphosphates (dNTPs) tothe primed circle result in the extension of each primer. Displacementof the newly synthesized strands result from elongation of the primerbehind it. Secondary priming events can subsequently occur on thedisplaced product strands of the initial RCA step.

The mutant may also be used in other quantitative amplification methodsin the art.

Kits are described herein that include a DNA polymerase variant asdescribed herein. Additional polymerases and other enzymes may beincluded in the kit. The DNA polymerase may be in a storage buffer(which may contain glycerol). A reaction buffer may be included whichmay be in concentrated form, and the buffer may contain additives (e.g.glycerol), salt (e.g. KCl), reducing agent, EDTA or detergents, etc. Thekit may further comprise deoxyribose nucleoside triphosphates e.g. one,two, three of all four of dATP, dTTP, dGTP and dCTP and/or one or moremodified nucleotides. The kit may optionally comprise one or moreprimers. The components of the kit may be combined in one container fora single step reaction, or one or more components may be contained inone container and separated from other components for sequential use orparallel use. The kit may also contain other reagents described abovethat may be employed in the method depending on how the method is goingto be implemented.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by referenceincluding U.S. application Ser. No. 16/598,554, filed Oct. 10, 2019, andU.S. Provisional Application No. 62/743,718, filed Oct. 10, 2018.

EXAMPLES Example 1: Multiply Primed Rolling Circle Amplification(MP-RCA)

Wild-type Phi29 DNA polymerase has been extensively used in multiplyprimed rolling circle amplification (MP-RCA) reaction under a lowreaction temperatures such as 30° C.-34° C. (de Vega, et al., PNAS, 107,38:16506-16511 (2010); Dean, et al., Genome Research, 11, 6:1095-1099(2001)). To characterize the thermostability of the bacteriophage DNApolymerase mutant, MP-RCA was performed at different temperatures andcompared with wild-type Phi29 polymerase. 50 μM of random 6 base primerthat contained two phosphorothioate linkages at the 3′ end and 1 ng ofpUC19 template were first annealed by heating to 95° C. for 3 minutes ina buffer (37 mM Tris, 50 mM KCl, 5 mM (NEUhSCU, 10 mM MgCl2, 0.025%Tween 20, pH 7.5) and subsequently cooled down to room temperature.Equivalent amounts of either wild-type or mutant polymerase was addedinto the reaction mixture and incubated at various times up to 4 hoursat different reaction temperatures ranging from 30° C. to 42° C. Thereaction mixture was then heated for 10 minutes at 65° C. to inactivatethe polymerases. The real-time monitoring of the MP-RCA reaction wasperformed by adding 1 μM of double stranded DNA binding dye SYTO®9(Molecular Probes, Eugene, Oreg.)) into the reaction mixture. Thefluorescence of the dye was detected using a CFX96 Touch™ Real-Time PCRdetection system (Bio-Rad, Hercules, Calif.) (FIG. 1A-1B). Theamplification product was then cleaved into fragments by Hindlllrestriction endonuclease (New England Biolabs, Ipswich, Mass.) andanalyzed in TBE agarose gel (FIG. 2A-2B).

The DNA polymerase mutant generated more amplification products under abroad range of reaction temperature (e.g. 30° C.-42° C.) as relative towild-type Phi29 polymerase (FIG. 2A, FIG. 2B). While the polymeraseactivity of the wild-type significantly decreased when the reactiontemperature was above 37° C., it was unexpected to find the mutantshowed increased reaction rate for DNA amplification when the reactiontemperature elevated.

As shown in FIG. 2A-2B, for example, the reaction rate was at least15-fold higher during the first 50 minutes of the reaction at elevatedtemperatures (e.g. 37.6° C. and 42.0° C.) than the otherwise identicalreaction at a lower temperature (e.g. 30° C.).

Example 2: Whole Genome Amplification (WGA)

WGA reaction was performed using 1 ng genomic DNA from HeLa cells astemplate. The reaction condition was otherwise identical to MP-RCA asdescribed above.

The real-time monitoring of the WGA reaction (FIG. 1C-1D) showed ahigher amplification efficiency when using the bacteriophage DNApolymerase mutant over a broad range of reaction temperatures (e.g. 30°C. to 41.5° C.). For example, during the first 50 minutes of the WGAreaction at 30° C., the mutant generated at least 1-fold moreamplification product as relative to wild-type Phi29 polymerase, whichfurther increased to over 2-fold at 37.3° C. At a temperature of 41.5°C., the wild-type Phi29 polymerase had completely lost any detectableenzymatic activity, while the mutant still showed high efficiency ofamplification.

As shown in FIG. 3A-3B, the bacteriophage DNA polymerase mutantgenerated at least 2-fold more amplification products as relative towild-type Phi29 polymerase after a 4-hour incubation which time framewas selected as the plateau of amplification yield as illustrated inFIG. 1A-1D. The yield increase was even more significant with thetemperature elevation, for example, to at least 9-fold above 35° C. Incontrast, the amplification yield when using wild-type Phi29 polymerasedecreased over four-fold when the temperature was elevated from 30° C.to 37.3° C., and there was no amplification product generated at 41.5°C.

Evenness of amplification is shown in FIG. 3C where the quantity ofspecific DNA sequences was measured with quantitative PCR at 4 distinctchromosomal locations (3p, 4p, 7p, 13p). The quantity of the DNA fromeach loci was compared to an unamplified control DNA standard andnormalized for the amount of amplified DNA product (FIG. 3D). A value of1 (y-axis) indicates perfectly even amplification and 0 a failure toamplify. As shown the mutant enzyme produced DNA closer to evenamplification at all 4 loci, whereas products from wild-type phi29showed very low amplification of the specified regions despiteproduction of significant quantities of DNA. The wild-type productstherefore represent less-even amplification and/or production ofnonspecific, template-independent DNA.

The template-independent DNA amplification was assessed by performingthe otherwise identical reaction in the absence of template gDNA andmeasuring the product generated by nonspecific primer-directed DNAsynthesis (FIG. 4). The accumulation of these template-independentproducts may severely decrease the specificity of WGA reaction. Theability of the mutant polymerase to perform WGA at elevated (42° C.)temperature not only facilitates higher yield and more even coverage asdescribed but reduces the level on nonspecific amplification (FIG. 4)with random hexamer primers in conditions above.

Example 3: Next-Generation Sequencing (NGS) Analysis Using IlluminaSequencing

DNA of 3 genomes (R. palustris, E. coli, H. influenzae) varying in GCcontent was mixed and used as template for WGA. The WGA was performed at41.5° C. (mutant) for 4 hours or as recommended by the commercial WGAformulations including wild-type (Illustra® Single-Cell GenomiPhi DNAAmplification Kit (GE Healthcare, Life Sciences, Marlborough, Mass.),REPLI-g® Single Cell Kit (Qiagen, Germantown, Md.) TruePrime® (Expedeon,San Diego, Calif.)) or different mutant (EquiPhi™ Phi29 (ThermoFisherScientific, Waltham, Mass.)) phi29 DNA polymerases. Reactions contained100 pg input of the mixed genomic template DNA and incubated for 2 hoursor as recommended. The WGA product was then purified and prepared forIllumina NextSeq® sequencing (Illumina, San Diego, Calif.) followingstandard protocol. Unamplified DNA was also sequenced as a control. Thepercentage of reads that mapped to each individual genome was calculatedand normalized to the unamplified sample reads, where 1 equals an exactmatch to the unamplified sample, a value >1 indicates over-amplificationand <1 under amplification.

These results are shown in Table 1 below and indicate a more equalamplification of all 3 genomes by the Mutant 1 DNA polymerase ascompared to the alternative WGA formulations.

TABLE 1 Demonstration of improved even coverage of 3 amplified genomescompared to the alternative WGA formulations using commercial Phi29polymerases using an Illumina NextSeq platform. H. inf E. Coli R.Palustris WGA Polymerase (38% GC) (50% GC) (65% GC) Unamplified 1 1 1MUTANT 1 1.9 1.1 0.51 Thermo EquiPhi (mut.) 2.1 1.8 0.029 GE-SC (wt)0.54 2.02 0.69 True Prime (wt) 4.1 0.00054 0.000051 Qiagen SC (wt) 3.20.84 0.0025

To analyze with complex (human genomic) DNA, Illumina sequencing readsusing libraries prepared from WGA reactions using the indicated WGApolymerase and human gDNA show even coverage (FIG. 5). 100 ng of DNAfrom each reaction was converted to Illumina libraries using theNEBNext® Ultra™ II Kit (New England Biolabs, Ipswich, Mass.) withbarcoded adaptors, and sequenced on a NextSeq instrument with reads downsampled to an equivalent number for each amplification method. Thegenome was separated into 100 bp regions and the GC percentagecalculated for each, and the number of reads representing each regiongraphed relative to its natural frequency in the genome (FIG. 5); i.e.perfect coverage of every region results in a straight line at y=1 onthe graph. As shown, an unamplified library made from pure genomic DNA(100 ng) results in the flattest profile, with using two examples ofmutant polymerases described herein producing similarly even (flat)coverage. Commercial WGA formulations using wild-type Phi29 andrecommended conditions (illustra Single-Cell GenomiPhi DNA AmplificationKit, REPLI-g Single Cell Kit, TruePrime) as well as a different mutantpolymerase at elevated temperature (EquiPhi, 45° C.) produce more unevencoverage as seen with overamplified (>1) and underamplified (<1)regions.

Example 4: Analysis by Oxford Nanopore Sequencing

DNA of 4 genomes (R. palustris, E. coli, H. influenza, P. falciparum)varying in GC content was mixed at certain ratio and used as templatefor WGA. The WGA was performed with 100 pg input of the mixed genomictemplate DNA and incubated for 2 hours (wild-type, 30° C.; and mutantPhi29, 42° C.) or as instructed by manufacturer (Qiagen and GE). The WGAproduct was then prepared for Oxford Nanopore MinION sequencing (OxfordNanopore Technologies, Oxford, UK) following standard protocols. Thenumber of sequencing reads from each sample is shown in Table 2 below,with the number containing chimeric sequence (fusion of non-consecutiveor opposite strand template sequence) and the percentage of chimericreads out of the total indicated Table 2.

TABLE 2 WGA Polymerase Chimeric Standard % Chimeric Qiagen SC (wt)45,943 68,315 40.2 GE V3 (wt) 142,873 219,883 39.4 WT Phi29 39,43043,290 47.7 MUTANT 1 36,940 291,413 11.2

A similar analysis is shown in Table 3 wherein 100 pg of human (HeLa)genomic DNA was used as template for amplification. Mutant reaction wasincubated for 2 hours at 42° C. or as recommended by the manufacturer.The percentage of reads containing chimeric DNA sequence is shown belowand again indicates a reduced frequency of chimera formation with themutant polymerase.

TABLE 3 WGA Polymerase Chimeric Standard % Chimeric Unamplified 19439276 0.49% MUTANT 3,893 22,117   15% Thermo EquiPhi (mut.) 10,80231,977   25% GE-HY (wt) 8,491 18,282   32% True Prime (wt) 5,223 23,065  18% Qiagen SC (wt) 7,053 18,610   27%

These results show that the DNA polymerase variant at 42° C. producessignificantly fewer chimeric DNA products as compared to wild-type Phi29and standard WGA methods.

Example 5: Improved Error Rate of Mutant Phi29 Polymerase Using PacBioSequencing

DNA products were produced using a restriction enzyme-digested M13 DNAsubstrate using either wild-type or mutant Phi29 in a low salt (50 mMK-Acetate) or high salt (215 mM K-Acetate) buffer. DNA was prepared forPacific Biosciences (Menlo Park, Calif.) RSII sequencing and the secondstrand error rate of the DNA polymerase calculated as described in(Potapov et, al., Nucleic Acids Res 46(11):5753-5763 (2018)). In bothbuffer conditions the error rate of the Mutant Phi29 was ˜2-fold lowerversus the wild-type Phi29, showing that the reduction in error rateassociated with the ability to perform reactions at 42° C. efficientlyis a property of the DNA polymerase variant and not the bufferconditions.

TABLE 4 Low salt error High salt error Polymerase rate (×10{circumflexover ( )} − 6) rate (×10{circumflex over ( )} − 6) Wild-type Phi29 15 17Mutant 1 9.2 8.6

SEQUENCE LISTING SEQ ID NO: 1MPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK SEQ ID NO: 2MPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHAERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRRAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGAEPVELYLTNVDLELIQEHYEMYNVEYIDGFKFREKTGLFKEFIDKWTYVKTHEKGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYAKEVDGKLIECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFRVGFSSTGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 3MSRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEQHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHTERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRKAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGVEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKDFIDKWTYVKTHEEGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLKDDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYVKEVDGKLKECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFAVGFSSMGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 4MSRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEQHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYRGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHTERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRKAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGVEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKDFIDKWTYVKTHEEGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLKDDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYVKEVDGKLKECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFAVGFSSMGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 5 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVWLERHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHKERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRYAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGGEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKDFIDKWTYVKTHEEGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFKVGFSSKGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 6 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHNGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHKERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRYAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGGEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKDFIDKWTYVKTHEEGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFKVGFSRSGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 7 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHKERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRGAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGEEPVELYLTNVDLELIQEHYEVYNVEYIDGFKFREKTGLFKNFIDKWTYVKTHENGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYKKEVDGKLGECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFQVGFSSNGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 8 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHIERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRQAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGTEPVELYLTNVDLELIQEHYEIYNVEYIDGFKFREKTGLFKTFIDKWTYVKTHENGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYKKEVDGKLRECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFQVGFSSKGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 9 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHEERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRQAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGGEPVELYLTNVDLELIQEHYEIYNVEYIDGFKFREKTGLFKTFIDKWTYVKTHETGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYGKEVDGKLDECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFKVGFSSKGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 10 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHGERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRHAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGNEPVELYLTNVDLELIQEHYEIYNVEYIDGFKFREKTGLFKTFIDKWTYVKTHENGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYKKEVDGKLRECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFEVGFSSDGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 11 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHKERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRHAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGDEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKNFIDKWTYVKTHETGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYKKEVDGKLGECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFDVGFSSSGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 12 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHGERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRHAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGNEPVELYLTNVDLELIQEHYEIYNVEYIDGFKFREKTGLFKTFIDKWTYVKTHEEGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLRECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFEVGFSSSGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 13 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHYERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRGAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGDEPVELYLTNVDLELIQEHYEVYNVEYIDGFKFREKTGLFKNFIDKWTYVKTHETGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYKKEVDGKLVECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFKVGFSSEGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 14 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHYERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRGAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGTEPVELYLTNVDLELIQEHYEIYNVEYIDGFKFREKTGLFKNFIDKWTYVKTHETGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYKKEVDGKLRECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFHVGFSSKGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 15 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHGERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIREAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGTEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKNFIDKWTYVKTHENGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYKKEVDGKLRECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFHVGFSSKGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 16 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHKERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRAAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGEEPVELYLTNVDLELIQEHYEIYNVEYIDGFKFREKTGLFKQFIDKWTYVKTHEEGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLGECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFDVGFSSKGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 17 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHYERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRQAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGDEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKDFIDKWTYVKTHETGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYGKEVDGKLVECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFDVGFSSDGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 18 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHYERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRAAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGEEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKNFIDKWTYVKTHEEGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFNVGFSSNGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 19 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHIERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRHAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGGEPVELYLTNVDLELIQEHYEVYNVEYIDGFKFREKTGLFKNFIDKWTYVKTHENGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLGECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFKVGFSSNGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 20 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHIERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRAAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGGEPVELYLTNVDLELIQEHYEIYNVEYIDGFKFREKTGLFKTFIDKWTYVKTHENGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLDECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFNVGFSSSGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 21 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHGERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRHAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGDEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKNFIDKWTYVKTHENGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLGECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFQVGFSSDGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 22 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHIERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRYAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGDEPVELYLTNVDLELIQEHYEVYNVEYIDGFKFREKTGLFKNFIDKWTYVKTHETGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLDECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFHVGFSSNGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 23 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHIERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRGAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGEEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKTFIDKWTYVKTHETGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYGKEVDGKLGECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFEVGFSSNGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 24 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHEERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRHAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGGEPVELYLTNVDLELIQEHYEVYNVEYIDGFKFREKTGLFKDFIDKWTYVKTHETGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYGKEVDGKLDECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFDVGFSSDGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 25 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHEERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRAAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGTEPVELYLTNVDLELIQEHYEIYNVEYIDGFKFREKTGLFKNFIDKWTYVKTHENGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYGKEVDGKLDECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFQVGFSSNGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 26 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHIERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRNAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGTEPVELYLTNVDLELIQEHYEVYNVEYIDGFKFREKTGLFKNFIDKWTYVKTHETGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYGKEVDGKLVECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFEVGFSSKGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 27 MutantMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHEERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRHAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGDEPVELYLTNVDLELIQEHYEVYNVEYIDGFKFREKTGLFKTFIDKWTYVKTHETGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFNVGFSSEGKPKPVQVNGGVVLVDSVFTIK

What is claimed is:
 1. A method, comprising: combining with a targetDNA, a DNA polymerase with an amino acid sequence that has at least 99%sequence identity with SEQ ID NO:5; and amplifying the target DNA. 2.The method according to claim 1, further comprising amplifying thetarget DNA in a high salt buffer.
 3. The method according to claim 1,further comprising amplifying the target DNA at a temperature in therange of 30° C.-42° C.
 4. The method according to claim 3, furthercomprising obtaining reduced sequence bias of the substrate DNA with thepolymerase in claim 1 compared to the wild-type Phi29 DNA polymeraseunder the same reaction conditions.
 5. The method according claim 3,further comprising obtaining a higher rate of amplification compared tothe rate of amplification using wild-type Phi29 DNA polymerase under thesame reaction conditions.
 6. The method according to claim 3, furthercomprising obtaining an improved amplification yield after a reactiontime of 2 hours compared to the yield obtained with wild-type Phi29 DNApolymerase under the same reaction conditions.
 7. The method accordingto claim 1, further comprising amplifying the target DNA in a high saltbuffer.
 8. A method, comprising: combining with a target DNA, a DNApolymerase with an amino acid sequence that has at least 95% sequenceidentity with SEQ ID NO:2 and has at least one, two, three, four, five,six or seven amino acid substitutions at positions selected from thegroup consisting of: 147, 221, 318, 503, 511, 544 and 550; andamplifying the target DNA.
 9. The method of claim 8, wherein the one toseven substitutions are selected from the group of substitutionsconsisting of: A147K, R221Y, A318G, A503M, I511V, R544K and T550K. 10.The method according to claim 8, further comprising amplifying thetarget DNA at a temperature in the range of 30° C.-42° C.
 11. The methodaccording to claim 8, further comprising obtaining an improvedrepresentation of the substrate DNA compared to the biasedrepresentation of wild-type Phi29 DNA polymerase under the same reactionconditions.
 12. The method according claim 8, further comprisingobtaining a higher rate of amplification compared to the rate ofamplification using wild-type Phi29 DNA polymerase under the samereaction conditions.
 13. The method according to claim 8, furthercomprising obtaining an improved yield of amplification after a reactiontime of 2 hours compared to the yield obtained with wild-type Phi29 DNApolymerase under the same reaction conditions.